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. 2020 Aug 31;217(11):e20191688. doi: 10.1084/jem.20191688

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© 2020 Helfricht et al.

This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).

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Figure 2. — ZBTB24 promotes DSB repair via c-NHEJ. (A) Schematic representation of the EJ5-GFP reporter for NHEJ. (B) HEK293T EJ5-GFP cells were treated with the indicated siRNAs and, 48 h later, cotransfected with I-SceI (pCBASce) and mCherry expression vectors. The ratio of GFP/mCherry-expressing cells was counted by flow cytometry 48 h later. The mean ± SD of two independent experiments is shown. Statistical significance was calculated using Student’s t test (*, P < 0.05). (C) Cells from B were subjected to RNA extraction. cDNA was synthesized from total RNA samples followed by qPCR to determine the expression levels of ZBTB24. The mean ± SEM of two independent experiments is shown. Statistical significance was calculated using Student’s t test (**, P < 0.01; ***, P < 0.001). (D) Schematic of the plasmid integration assay. pEGFP-C1 plasmid containing Neo and GFP markers is linearized with the indicated restriction enzymes and transfected into U2OS cells. Stable integrants are selected on medium containing G418. GFP was used as a control for transfection efficiency. (E) Plasmid integration assays in U2OS cells transfected with the indicated siRNAs. The mean ± SEM of two to four independent experiments is shown. Statistical significance was calculated using Student’s t test (**, P < 0.01; ****, P < 0.0001). (F) As in C, except that cells from E were used. The mean ± SEM of two independent experiments is shown. Statistical significance was calculated using Student’s t test (**, P < 0.01; ***, P < 0.001). (G) VH10-SV40 cells were treated with the indicated siRNAs for 48 h, exposed to different doses of IR, and scored for clonogenic survival. The mean ± SEM of two independent experiments is shown. (H) As in C, except that cells from G were used to monitor XRCC4 expression. The mean ± SEM of two independent experiments is shown. Statistical significance was calculated using Student’s t test (***, P < 0.001). (I) As in C, except that cells from G were used. The mean ± SEM of two independent experiments is shown. Statistical significance was calculated using Student’s t test (***, P < 0.001).